Angiotensin II (Ang II) plays an essential role in maintaining body sodium and fluid balance and normal blood pressure by regulating proximal tubular sodium reabsorption. Ang II exerts powerful effects on sodium transport and cell growth by activating cell surface receptors on brush borders and basolateral membranes of proximal tubule cells. However, we have new evidence that a) extracellular Ang II is taken up by proximal tubule cells in vivo and in vitro; b) microinjection of Ang II directly into the cells increases intracellular calcium; and c) Ang II induces RNA transcription and expression in isolated nuclei. Our results suggest that internalized Ang II may act as an intracellular hormone to play important physiological and pathological roles in these cells. In this project, we propose to test the general hypothesis that extracellular Ang II is taken up by proximal tubule cells through AT1A receptor-mediated internalization, and that internalized Ang II binds to cytoplasmic and nuclear receptors to induce intracellular responses. To test this hypothesis, we will conduct both in vitro and in vivo studies, using complementary biochemical, morphological, cellular and molecular biology approaches. In Aim I, we will study whether proximal tubular cells take up extracellular Ang II in vitro and in vivo and elucidate the mechanisms by which Ang II receptors and the endocytotic machinery regulate Ang II trafficking. In Aim II, we will use confocal microscopy, state-of-the-art EM autoradiography and immunohistochemistry to trace intracellular trafficking pathways of internalized Ang II to the endosomal compartments and its translocaUon to the nucleus in vitro and in vivo. In Aim III we will study whether microinjection of Ang II increases intracellular calcium through activation of cytoplasmic AT1 receptors and the cellular mechanisms involved. Finally, in Aim IV we will investigate whether internalized Ang II binds to intracellular Ang II receptors to activate transcription factor NFkappaB and its translocation to the nucleus, and whether internalized Ang II stimulates nuclear receptors to increase transcription and expression of the Na+/H+ exchanger NHE3 and pro-inflammatory cytokines. These studies will provide new insights into the important role of internalized Ang II in renal physiology and Ang II-induced hypertensive renal injury.